CN210419994U

CN210419994U - Cell culture device

Info

Publication number: CN210419994U
Application number: CN201920716975.2U
Authority: CN
Inventors: 黄楠; 薛勇; 于涵; 涂秋芬; 杨志禄
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-04-28
Anticipated expiration: 2029-05-17

Abstract

The present application relates to the field of cell culture environment construction, and specifically relates to a cell culture apparatus comprising a culture implement and: the transportation pipeline is communicated with the culture device head and tail to form a circulation loop; a driving device connected with the conveying pipeline and used for driving the culture medium to flow out of the culture device and flow back to the culture device through the circulating loop; and the air supply device is communicated with the conveying pipeline and is arranged at the upstream of the driving device, and an air outlet pipe is arranged at the downstream of the conveying pipeline. Through making the culture medium along circulation loop circulation operation, utilize air feeder and outlet duct cooperation to add the required material of culture medium, make the culture medium constantly increase the content of this kind of interpolation material at the in-process of constantly circulating to constantly keep the state rich in this material, thereby construct stable culture medium environment, in order to guarantee cell or tissue culture.

Description

Cell culture device

Technical Field

The application relates to the field of cell culture environment construction, in particular to a cell culture device.

Background

The construction of a stable culture medium environment is an important condition for ensuring smooth development of some experiments and production, and taking the construction of a stable hydrogen-rich culture medium environment as an example, the stable hydrogen-rich culture medium is a basis for the development of cytology and histology experiments in the field of nano physical therapy gas biomedicine. At present, the main mode of preparing the hydrogen-rich culture medium is to mix sterile oxygen-rich water and a high-concentration culture medium according to a certain proportion, the preparation mode is convenient and simple, the hydrogen-rich culture medium can be prepared and used immediately, but the hydrogen concentration can be rapidly reduced along with the use of the hydrogen-rich culture medium, so that the hydrogen concentration in the culture medium is unstable and the culture effect is poor, in order to maintain the hydrogen concentration, the culture medium needs to be repeatedly replaced in a short time, the risk of infecting mixed bacteria can be greatly increased in the replacement process, and the resource waste can be caused.

SUMMERY OF THE UTILITY MODEL

The present application aims to provide a cell culture apparatus to solve the problem of constructing a stable medium environment.

The embodiment of the application is realized as follows:

in one aspect, embodiments of the present application provide a cell culture apparatus, including a culture device, further including:

the transportation pipeline is communicated with the culture device head and tail to form a circulation loop;

a driving device connected with the conveying pipeline and used for driving the culture medium to flow out of the culture device and flow back to the culture device through the circulating loop;

and the air supply device is communicated with the conveying pipeline and is arranged at the upstream of the driving device, and an air outlet pipe is arranged at the downstream of the conveying pipeline.

The utility model provides a transport pipe way that sets up end to end connection culture apparatus forms circulation circuit, makes the culture medium follow circulation circuit and move in cycles through drive arrangement, links into transport pipe way with air feeder on the circulation path of culture medium, makes gaseous and culture medium on the way mix to increase the concentration of this kind of gaseous matter in the culture medium or its contained element, and set up the outlet duct on the transport pipe way of circulation path low reaches, will not dissolved gas outgoing before the culture medium gets back to culture apparatus.

Through making the culture medium along circulation loop circulation operation, utilize air feeder and outlet duct cooperation to add the required material of culture medium, make the culture medium constantly increase the content of this kind of interpolation material at the in-process of constantly circulating to constantly keep the state rich in this material, thereby construct stable culture medium environment, in order to guarantee cell or tissue culture. The application provides a cell culture device can guarantee that required material can not consume along with the culture process, need not to change the culture medium repeatedly and guarantees that the content is stable, has reduced the chance that the culture medium exposes, and whole culture process is in comparatively confined environment, and the risk of infecting miscellaneous fungus is little, has also reduced the culture medium extravagant.

In an embodiment of the application, further, a first gas filter is provided on a passage between the gas supply device and the transport pipe. The first gas filter can play a role in filtering bacteria and isolating bacteria, so that external bacteria are prevented from being mixed into gas from the gas supply device and entering the circulation loop, and the sterile operation of the circulation loop is further ensured.

In an embodiment of the present application, further, an air flow adjusting valve is disposed on a path between the air supply device and the transportation pipeline. The air flow regulating valve is arranged on the passage between the air supply device and the conveying pipeline, so that the air flow can be conveniently regulated according to the required concentration, the concentration of the required substances in the culture medium can be controlled, a stable culture medium environment can be constructed, and the corresponding substance concentrations can be provided at different stages of culture; before the culture medium circulation needs to be suspended or stopped, the air flow regulating valve can also block the passage where the culture medium circulation is positioned, so that part of the culture medium is prevented from flowing into the air supply device when the culture medium circulation is stopped.

In one embodiment of the present application, further, the first gas filter is disposed between the gas flow regulating valve and the gas supply device.

In an embodiment of the present application, further, a gas-liquid mixer disposed on the transport pipeline is further included; the gas-liquid mixer comprises a mixing cavity, and a first pipe section, a second pipe section and a third pipe section which extend outwards from the mixing cavity:

the first pipe section is connected with the upstream section of the transportation pipeline;

the second pipe section is communicated with the gas supply device;

the third pipe section is connected with the downstream section of the conveying pipeline, which is provided with the driving device.

Through setting up the gas-liquid mixer, increase a mixing chamber on circulation loop, the culture medium gets into mixing chamber from first pipeline section, and gaseous second pipeline section entering mixing chamber, culture medium and gaseous in mixing chamber after the outflow from the third body, mixing chamber provides the space that allows the mixture of gaseous and culture medium offset, makes gaseous and culture medium mix more evenly.

In an embodiment of the application, further, a gas-liquid separator is arranged on the conveying pipeline between the driving device and the culture device, the gas-liquid separator comprises a separation cavity, the separation cavity is provided with a liquid outlet pipe communicated with the culture device through the conveying pipeline, and the gas outlet pipe is connected with the separation cavity. The gas-liquid separator provides a separation chamber for buffering the culture medium mixed with gas in the separation chamber, so that bubbles formed by undissolved gas rise and separate from the culture medium and are finally discharged from the gas outlet pipe, and the culture medium separated from bubbles flows out of the liquid outlet pipe and returns to the culture device. Since the gas supplied from the gas supply means may not be a substance in the gas atmosphere required for cell growth, bubbles in the culture vessel are further reduced by the gas-liquid separator, so that the culture medium and the culture vessel do not contain excessive gas added, thereby maintaining the gas atmosphere required for cell growth.

In an embodiment of the present application, further, a liquid flow regulating valve is disposed on the passage of the liquid outlet pipe. The liquid flow regulating valve can regulate the circulation speed of the culture medium on the whole circulation loop; secondly, the flow rate of the culture medium flowing back to the culture device through the liquid outlet pipe can be instantly adjusted, and the amount of the culture medium cached in the gas-liquid separator is controlled, so that the culture medium flowing into the separation cavity has enough time to separate bubbles.

In an embodiment of the present application, further, a second gas filter is disposed on the path of the outlet pipe. The second gas filter can play the role of filtering and isolating bacteria, prevent external bacteria from reversely entering the separation cavity from the gas outlet pipe and mixing with the culture medium to pollute the whole circulation loop, and particularly further ensure the sterile operation of the circulation loop under the condition that the gas outlet flow of the gas outlet pipe is small.

In an embodiment of the present application, further, the containing cavity of the culture device for containing the culture medium is provided with an opening with a sealing cover body, the cover body is respectively provided with an output pipe and an inlet pipe, the output pipe is communicated with the upstream section of the transportation pipeline, the inlet pipe is communicated with the downstream section of the transportation pipeline, and the output pipe extends to the bottom of the containing cavity. Lid and output tube, input tube can set up to whole, conveniently open the holding chamber, still conveniently insert the transportation pipeline with the culture apparatus, and the output tube stretches into the bottom of holding chamber and is favorable to discharging the culture medium of the deepest in holding chamber, makes the culture medium whole circulation in the holding intracavity get up, further keeps in the culture medium that material concentration is stable and even.

In an embodiment of the application, further, the drive device comprises a peristaltic pump, and the portion of the transport conduit passing through at least the peristaltic pump is a hose. The peristaltic pump drives the circulation of the internal culture medium in a mode of extruding the hose, and the culture medium and the gas are extruded and mixed again when flowing through the peristaltic pump after being mixed, so that the mixing effect of the culture medium and the gas is enhanced, and the gas dissolution rate is favorably improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a schematic structural diagram of a cell culture apparatus according to an embodiment of the present application.

Icon: 1-a culture device; 12-a cover body; 21-an output pipe; 22-an inlet tube; 3-gas-liquid mixer; 31-a first tube section; 32-a second tube section; 33-a third tube section; 4-a gas supply device; 41-air supply cannula; 42-a first gas filter; 43-gas flow regulating valve; 5-a drive device; 51-a power supply; 6-gas-liquid separator; 61-a liquid flow regulating valve; 62-a second gas filter; 63-a liquid outlet pipe; and 64-an air outlet pipe.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present application, presented in the accompanying drawings, is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present application, it should be noted that if the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are used for indicating the orientation or positional relationship based on the orientation or positional relationship shown in the drawings or the orientation or positional relationship which is usually placed when the product of the application is used, the description is only for convenience and simplicity, and the indication or suggestion that the referred device or element must have a specific orientation, be constructed in a specific orientation and be operated, and thus, should not be construed as limiting the present application. Furthermore, the appearances of the terms "first," "second," and the like in the description herein are only used for distinguishing between similar elements and are not intended to be construed as indicating or implying relative importance.

Furthermore, the terms "horizontal", "vertical" and the like when used in the description of the present application do not require that the components be absolutely horizontal or overhanging, but may be slightly inclined. For example, "horizontal" merely means that the direction is more horizontal than "vertical" and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.

In the description of the present application, it should also be noted that, unless otherwise explicitly stated or limited, the terms "disposed," "mounted," "connected," and "connected" should be interpreted broadly, e.g., as being fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art.

Examples

The application provides a cell culture device for cell culture, it can provide stable culture medium environment, need not to change the culture medium repeatedly, reduces and makes culture apparatus 1 expose the risk of infecting outside miscellaneous fungus in the outside air, improves accuracy and the success rate of experiment, production.

Referring to fig. 1, the cell culture apparatus includes a culture device 1, the culture device 1 is provided with a containing cavity for containing a culture medium and providing a cell culture space, and further includes a transport pipeline, a driving device 5 and a gas supply device 4.

The head and the tail of the transportation pipeline are connected with the culture device 1, and the head and the tail of the transportation pipeline are communicated with the accommodating cavity to form a circulation loop.

The above-mentioned circulation circuit is provided with a driving means 5, and the driving means 5 is used for operating the material flow on the circulation circuit, i.e., the culture medium in the culture device 1 is operated along the circulation circuit flow.

The gas supply means 4 is arranged upstream of the transport pipe, i.e. near the end of the culture medium that flows out of the culture device 1, so as to mix gas into the culture medium; and an outlet pipe 64 is provided downstream of the transport pipe, i.e., the outlet pipe 64 is provided near one end where the culture medium flows back to the culture device 1. Gas enters the circulation loop from the gas supply 4 to mix with the culture medium, part of the gas dissolves in the culture medium to increase the concentration of the substance in the culture medium, and undissolved gas travels to the gas outlet 64 and is discharged.

The whole circulation loop of the culture medium is closed off from the outside and is exhausted to the outside only at the outlet pipe 64, wherein the opening of the outlet pipe 64 may be directed to the upper part to facilitate the ascending and exhausting of the gas.

In this embodiment, for example, a stable hydrogen-rich culture medium is constructed, and the gas supply device 4 supplies hydrogen to the circulation loop, and the gas supply device 4 can be a balloon filled with pure hydrogen.

In this embodiment, the above-mentioned culture device 1 is a culture bottle, the interior of the culture bottle is a containing cavity, the culture bottle is provided with a bottle opening, the bottle opening is provided with a detachable and sealed cover 12, the cover 12 is provided with two through holes, an output pipe 21 and an inlet pipe 22 are respectively fixed in the two through holes, and the output pipe 21 extends to the bottom of the containing cavity. The outlet pipe 21 and the inlet pipe 22 are tightly connected and sealed with the through hole of the cover 12. The output pipe 21 and the input pipe 22 are respectively connected with the head end and the tail end of the transportation pipeline in a fixed connection mode or a detachable connection mode.

The outlet pipe 21 and the inlet pipe 22 in this embodiment are needle tubes, and silicone rubber rings are sleeved on the needle tubes to seal the through holes on the cap body 12. In other embodiments, culture device 1 can be any other container with a sealable holding chamber, and output tube 21 and inlet tube 22 can be other lines that allow the flow of culture medium. In this embodiment, the transportation pipeline is at least for having elastic silicone rubber tube end to end, and the silicone rubber tube cover at transportation pipeline both ends is established in the one end that the needle tubing exposes the holding chamber, and its position of connection is inseparable in order to guarantee sealedly when transportation pipeline is connected with the needle tubing, when needs are dismantled, with lid 12 twist off the isolated culture utensil 1 can, conveniently operate, disinfect respectively cultivation utensil 1 and transportation pipeline. The culture device 1 may be a disposable cell culture device, and may be replaced with a new disposable cell culture device after being separated.

The driving device 5 may be any type of power pump disposed on the path of the transportation pipeline as long as the culture medium can flow along the transportation pipeline, in this embodiment, the driving device 5 employs a peristaltic pump, at least a portion of the transportation pipeline passing through the peristaltic pump is a hose, and the peristaltic pump presses the hose to pump the culture medium inside the hose. In the pumping process, the culture medium and the peristaltic pump are separated by the hose, the sealing performance of the circulation loop is good, and the culture medium and the peristaltic pump are prevented from being polluted by each other. When the peristaltic pump extrudes the hose, larger bubbles are crushed into smaller bubbles, and the culture medium and the gas in the hose are disturbed and mixed again, so that the dissolution rate of the hydrogen is improved.

For better mixing of the culture medium and the hydrogen gas, a gas-liquid mixer 3 is arranged on the transport pipe, which gas-liquid mixer 3 comprises a mixing chamber, and three pipe sections extending outwards from the mixing chamber. Wherein the first pipe section 31 is connected to the upstream of the transport pipe, i.e. to the end close to the delivery pipe 21 of the culture device 1, and is communicated with the containing cavity of the culture device 1; the second pipe section 32 is in communication with the gas supply means 4; the third pipe section 33 is connected downstream of the transport pipe, i.e. close to the drive 5.

The air supply inlet of the air supply device 4 is provided with an air supply insertion tube 41, the air supply insertion tube 41 is inserted into one end of the second pipe section 32, and the air supply insertion tube 41 is tightly connected with the second pipe section 32 to prevent air leakage. Illustratively, the inner diameter of the second tube section 32 in the natural state is smaller than the outer diameter of the gas supply cannula 41, and the second tube section 32 is a silicone rubber tube having elasticity.

The driving device 5 provides power to make the culture medium enter the transportation pipeline through the output pipe 21 and enter the mixing cavity through the first pipe section 31, make hydrogen enter the mixing cavity from the gas supply device 4 through the second pipe section 32, after the culture medium and hydrogen are mixed in the mixing cavity, the culture medium flows out of the mixing cavity from the third pipe section 33 under the power of the driving device 5 and enters the transportation pipeline to continue flowing, and the undissolved gas is discharged through the gas outlet pipe 64 before the culture medium returns to the accommodating cavity of the culture device 1.

In order to further reduce the air bubbles contained in the culture medium returned to the holding chamber, so as to maintain the gas atmosphere required for cell culture in the holding chamber, the culture medium passes through the gas-liquid separator 6 before returning to the holding chamber. The gas-liquid separator 6 is disposed downstream of the transport pipe and on the path between the driving device 5 and the culture medium.

The gas-liquid separator 6 comprises a separation chamber as shown in FIG. 1, a liquid outlet pipe 63 is arranged in the separation chamber, the liquid outlet pipe 63 is communicated with the inlet pipe 22 of the culture apparatus 1 through a conveying pipeline, and the gas outlet pipe 64 is arranged in the separation chamber. To further facilitate circulation loop operation and venting, the inlet of outlet conduit 63 is near the bottom of the separation chamber and the inlet of outlet conduit 64 is near the top of the separation chamber. The culture medium mixed with the bubbles flows out of the mixing cavity, enters the separation cavity of the gas-liquid separator 6 after passing through the driving device 5, and is temporarily stored and buffered in the separation cavity, as shown in figure 1, the culture medium flows downwards to the bottom of the separation cavity under the action of gravity, the bubbles rise in the process of flowing downwards and temporarily storing in the separation cavity, and the gas rises to the top of the separation cavity after the bubbles break. After the culture medium is separated from the bubbles, the culture medium flows out of the liquid outlet pipe 63 and returns to the accommodating cavity through the inlet pipe 22, and the gas in the separation cavity is discharged from the gas outlet pipe 64.

Further, the gas outlet pipe 64 may be connected to a gas collecting device, such as a gas collecting bag, a gas storage bottle, etc., to recover the undissolved hydrogen gas for secondary use.

In order to further isolate the circulation loop from the isolated outside environment, a first gas filter 42 is provided on the passage of the gas supply 4 and the second tube section 32 to prevent external bacteria from entering the circulation loop through the gas supply 4; a second gas filter 62 is provided at the outlet pipe 64 to prevent external bacteria from entering the circulation loop through the outlet pipe 64.

In addition, an air flow adjusting valve 43 is disposed on the path of the air supply device 4 connected to the transportation pipeline (i.e. the path between the air supply device 4 and the second pipe section 32) so as to adjust the air flow of the supplied air. When the required hydrogen concentration is high, the gas flow regulating valve 43 is adjusted to increase the flow rate of the gas supplied by the gas supply device 4; when the required hydrogen concentration is small, the gas flow regulating valve 43 is adjusted to reduce the flow rate of the gas supplied from the gas supply device 4.

The gas flow regulating valve 43 also serves to prevent the culture medium from flowing into the passage to the gas supply means 4. When the culture apparatus provided in this embodiment is used, the driving unit 5 is first turned on to allow the culture medium to flow along the circulation path, and then the gas supply unit 4 and the gas flow regulating valve 43 are turned on to connect the gas supply path. Before the circulation is stopped, the air flow regulating valve 43 and the air supply device 4 are closed to block the air supply passage, so that when the circulation of the culture medium is stopped, the residual culture medium in the conveying pipeline flows into the air supply passage or the air supply device 4.

Alternatively, the first gas filter 42 is disposed between the gas flow regulating valve 43 and the gas supply device 4, and when the circulation is stopped, the first gas filter 42 is isolated from the culture medium, preventing the culture medium from being contaminated and inconvenient for secondary use.

The liquid outlet pipe 63 of the gas-liquid separator 6 is provided with a liquid flow regulating valve 61, and the liquid flow regulating valve 61 is used for regulating the flow quantity of the culture medium in the passage of the liquid outlet pipe 63. When the power of the driving device 5 is not changed, the circulation speed of the entire circulation circuit is decreased when the liquid flow regulating valve 61 is closed, and the circulation speed of the entire circulation circuit is increased when the liquid flow regulating valve 61 is opened.

In addition, the liquid flow regulating valve 61 can instantaneously regulate the amount of the liquid flowing out of the separation chamber and returning to the holding chamber of the culture apparatus 1 through the liquid outlet pipe 63. When the liquid flow regulating valve 61 is closed, the outflow volume of the separation cavity is immediately reduced, and the inflow volume can be reduced to be the same as the outflow volume after a period of time, so that the culture medium temporarily stored in the separation cavity is increased, the cache time of the culture medium flowing into the separation cavity is prolonged, and enough time is provided for separating bubbles included in the culture medium, so that the gas-liquid separation is more thorough. When the liquid flow regulating valve 61 is opened, the culture medium liquid temporarily stored in the separation cavity can be quickly released, and after the circulation of the culture medium is stopped, the culture medium in the separation cavity quickly flows into the accommodating cavity of the culture device 1.

The cell culture device provided by the embodiment enables the culture medium to run along the circulation loop, and hydrogen is mixed and then separated in the circulation process, so that part of hydrogen is dissolved in the culture medium, and the culture medium is rich in hydrogen. The cell culture apparatus provided in this embodiment can be used not only to enrich the medium with hydrogen, but also to add other substances to the medium, for example, to increase the oxygen content of the medium.

The medium is circulated and the material is added during the circulation, so that the medium is kept in a state of being rich in the added material.

In order to ensure that the cell culture device is kept clean and sterile, the cell culture device needs to be sterilized before use. Wherein, the cell culture apparatus 1, the gas-liquid mixer 3 and the gas-liquid separator 6 can be selected from high-temperature sterilization and high-temperature resistance glassware, and the whole conveying pipeline is made of medical silicon rubber.

The transport pipe in this embodiment is actually divided into a plurality of sections, including a first transport section, a second transport section and a third transport section. The first transport section connects the output pipe 21 of the culture device 1 and the first pipe section 31 of the gas-liquid mixer 3. The second transportation section connects the third pipe section 33 of the gas-liquid mixer 3 with the inlet of the separation chamber of the gas-liquid separator 6. The third transportation section connects the outlet pipe 63 of the gas-liquid separator 6 with the inlet pipe 22 of the culture device 1. Wherein the second transport section is connected to the driving means 5, i.e. in this embodiment, the second transport section passes the driving pump head of the peristaltic pump.

The cell culture apparatus provided in this example can enrich the culture medium with hydrogen by the following specific method:

after the cell culture device is sterilized and disinfected, a culture medium is added into the accommodating cavity of the culture device, then the cover body 12 of the accommodating cavity is closed, the output pipe 21 and the inlet pipe 22 on the cover body 12 are respectively connected with the conveying pipelines, the gas-liquid mixer 3 and the gas-liquid separator 6 are connected into the circulating loop along the preset circulating direction, then the gas supply device 4 is connected with the gas-liquid mixer 3, and the conveying pipeline between the gas-liquid mixer 3 and the gas-liquid separator 6 is connected into the driving pump head of the peristaltic pump. The device can be assembled on a sterile table after sterilization, wherein the disposable culture device 1, the mobile power supply 51, the detachable pump body 5 and the air supply device 4 are assembled after being sterilized by ultraviolet or alcohol.

Starting a circulation loop: opening the liquid flow regulating valve 61, starting the peristaltic pump, and allowing the circulation speed of the culture medium to be stable;

starting an adding path: the gas supply device 4 is started, the gas flow regulating valve 43 is opened, hydrogen enters the mixing cavity from the second pipe section 32, the hydrogen is mixed with the culture medium and then runs to the gas-liquid separator 6, part of the hydrogen is dissolved in the culture medium in the process, the hydrogen concentration in the culture medium is increased, the culture medium continuously circulates, the hydrogen concentration can be continuously increased, and therefore the culture medium is in a hydrogen-rich state.

The inventor finds through experiments that the hydrogen concentration of the culture medium can be adjusted and maintained at a certain concentration within the gradient range of 0.2-1.4 ppm by the cell culture device provided by the embodiment.

In actual use, the cell culture apparatus can be used to perform a variety of cytological or histological tests.

In this example, a human vascular endothelial cell line is taken as an example, and an adherent cell culture test is performed on the human vascular endothelial cell line, and the test method is as follows:

the cover body 12 with the outlet pipe 21 and the inlet pipe 22, the gas-liquid mixer 3, the gas-liquid separator 6 and the multi-section transportation pipeline for connection, which are sterilized at high temperature and high pressure, and the disposable incubator 1, the portable power source 51, the detachable pump body 5 and the gas supply device 4, which are sterilized by ultraviolet or alcohol, are taken out and connected in the connection manner as described above in this embodiment, and then the whole is placed in a sterilized clean bench.

The peristaltic pump is connected with a second transportation section of the transportation pipeline according to the preset circulation direction, namely the second transportation section is arranged at the position of a driving pump head of the peristaltic pump, the peristaltic direction is noticed during installation, and the reverse peristaltic direction is avoided.

Human vascular endothelial cell strain is recovered in DMEM-F12 medium containing 15% fetal bovine serum, centrifuged, added with 10ml of medium and resuspended. The holding chamber of the culture device 1 is then opened, the cell suspension is added to the holding chamber, and the chamber is closed with the lid 12.

The cell culture device (especially the components of the whole circulation loop: the culture device 1, the gas-liquid mixer 3, the gas-liquid separator 6 and the transport pipeline) is placed in an incubator, and after 6h, the adherence condition of the cells in the accommodating cavity of the culture device 1 is tested.

After the adherence is good, the peristaltic pump is connected with the movable power supply 51 and started, and then the gas flow regulating valve 43 is opened to regulate the gas flow so as to meet the concentration requirement required by culture.

When the culture medium needs to be changed, the air flow regulating valve 43 is closed first, and then the power supply 51 of the peristaltic pump is closed, so that the culture medium is prevented from being sucked back and flowing back to the air supply device 4. Then, the lid 12 is opened to pour out the waste liquid, the lid 12 is screwed after replacing the new culture medium, the power supply 51 is turned on again to start the peristaltic pump, and the air flow regulating valve 43 is opened.

When the experiment is finished and cells need to be collected, the air flow regulating valve 43 is closed, and then the power supply 51 is closed, so that the culture medium is prevented from being sucked back to the air supply device 4. Then, the waste liquid was collected into a sterilized centrifuge tube, the culture device 1 was washed with physiological saline, and then digested with trypsin, and then the culture medium was added to the new culture device 1, and the cells were separated into bottles.

The cell culture apparatus and the method for enriching the culture medium with hydrogen provided in this embodiment can also be conveniently used for studying the influence of hydrogen on the cell cycle level, and the following human vascular endothelial cell lines are taken as examples:

the normally passaged human endothelial cell line was subcultured into four flasks and then cultured in a conventional flask in DMEM-F12 medium containing 15% fetal bovine serum.

After 12h of culture, the cells were observed for logarithmic growth phase. Cells in the logarithmic growth phase were subjected to liquid exchange and serum starvation treatment using pure DMEM-F12 medium.

After serum starvation treatment is carried out for 24 hours, the cell culture device subjected to high-temperature and high-pressure sterilization is taken out, the cell culture device is assembled and connected according to the method and then placed in a sterilized ultra-clean workbench, and a peristaltic pump is connected with a second transportation section according to the preset circulation direction, so that the phenomenon that the peristaltic direction is wrong is avoided.

The starvation-treated medium was poured out and replaced with DMEM-F12 medium containing 15% fetal bovine serum, the newly replaced medium was added to the holding chamber of the culture device 1, the lid 12 was screwed on, the peristaltic pump was started, the air flow regulating valve 43 was opened, and the size of the air flow regulating valve 43 was adjusted to allow cells to develop under the predetermined hydrogen concentration condition.

After 6h of incubation, the air flow regulating valve 43 is closed and the peristaltic pump is then closed to prevent the back flow of the culture medium to the gas supply 4. Then, the waste liquid is collected into a sterilized centrifuge tube, the culture device 1 is washed by normal saline, then is digested by trypsin, and then the cells are blown down by a pipette tip, placed into a culture dish and blown off gently, and are centrifuged at 1000rpm for 3-5 minutes to precipitate the cells. And pouring out the supernatant, adding 4ml of 75% ethanol precooled by an ice bath, fixing for 1-12 h, carefully sucking out the supernatant, adding PBS (phosphate buffer solution), washing, centrifuging again for precipitation, and dyeing by using an propidium iodide staining solution in a dark place after precipitation. After incubation for 30 minutes in the absence of light, the cells were examined by flow cytometry.

In addition, the cell culture device can also adjust the size of the air flow adjusting valve 43 in real time so as to adjust the hydrogen concentration in the culture medium according to the test requirement, thereby meeting the real-time concentration requirements of different stages in the test process.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cell culture apparatus comprising a culture device, characterized by further comprising:

2. The cell culture apparatus of claim 1, wherein a first gas filter is provided in a passage between the gas supply means and the transport pipe.

3. The cell culture apparatus of claim 2, wherein a gas flow regulating valve is provided on a passage between the gas supply means and the transport pipe.

4. The cell culture apparatus of claim 3, wherein the first gas filter is disposed between a gas flow regulating valve and a gas supply.

5. The cell culture apparatus of any one of claims 1 to 4, further comprising a gas-liquid mixer disposed on the transport conduit; the gas-liquid mixer comprises a mixing cavity, and a first pipe section, a second pipe section and a third pipe section which extend outwards from the mixing cavity:

the second pipe section is communicated with the gas supply device;

6. The cell culture device according to claim 5, wherein a gas-liquid separator is arranged on the conveying pipeline between the driving device and the culture device, the gas-liquid separator comprises a separation cavity, the separation cavity is provided with a liquid outlet pipe communicated with the culture device through the conveying pipeline, and the gas outlet pipe is connected with the separation cavity.

7. The cell culture apparatus of claim 6, wherein the liquid outlet pipe is provided with a liquid flow regulating valve in its passage.

8. The cell culture apparatus of claim 1, wherein the passage of the gas outlet pipe is provided with a second gas filter.

9. The cell culture apparatus according to claim 1, wherein the chamber for accommodating the culture medium is provided with an opening with a sealing cover, the cover is provided with an outlet pipe and an inlet pipe, the outlet pipe is communicated with the upstream section of the transportation pipeline, the inlet pipe is communicated with the downstream section of the transportation pipeline, and the outlet pipe extends to the bottom of the chamber.

10. The cell culture apparatus of claim 1, wherein the drive device comprises a peristaltic pump, and the portion of the transport conduit passing through at least the peristaltic pump is a flexible tube.